ABSTRACT
On the basis of the linear killer plasmid pGKL1 from Kluyveromyces lactis, two new linear hybrid plasmids were constructed. One of these, pRSC126, carried the xylA gene from Streptomyces rubiginosus encoding the xylose isomerase. The other linear hybrid molecule, pRSC128, carried the hasB gene of Streptococcus pyogenes encoding the UDP glucose dehydrogenase. Construction was performed in a way that the putative cytoplasmic promoter element of ORF5 of pGKL2 was fused to the coding region of the heterologous genes. After transformation, in vivo recombination led to the establishment of linear hybrid vectors. Though efficiency of expression was low when compared with bacterial systems, cytoplasmic expression of both genes was clearly demonstrated.
Subject(s)
Aldose-Ketose Isomerases , Carbohydrate Epimerases/genetics , Gene Expression Regulation, Bacterial , Streptococcus pyogenes/genetics , Streptomyces/genetics , Uridine Diphosphate Glucose Dehydrogenase/genetics , Blotting, Southern , Carbohydrate Epimerases/metabolism , Chromosome Mapping , Cloning, Molecular , Genetic Vectors , Kluyveromyces/genetics , Open Reading Frames , Plasmids/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombination, Genetic , Transformation, Bacterial , Uridine Diphosphate Glucose Dehydrogenase/metabolismABSTRACT
Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.
Subject(s)
Gene Expression Regulation, Fungal , Kluyveromyces/genetics , Plasmids/genetics , Promoter Regions, Genetic , Bacillus megaterium/genetics , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , Cytoplasm , Escherichia coli , Genes, Reporter , Genetic Vectors , Glucose 1-Dehydrogenase , Glucose Dehydrogenases/genetics , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transcription, GeneticABSTRACT
Relative phylogenetic distances were estimated for those linear plasmids for which sequencing data were available by comparing the amino-acid sequences of the putative DNA- and RNA-polymerases, and phylogenetic trees were calculated. The relationships obtained accord well with those indicated by other structural characteristics of these genetic elements. It is obvious that linear plasmids constitute a separate group of genetic traits when compared with those of the adenoviruses. However, an overall relationship to these viruses is evident. Among the linear plasmids at least two main groups can be recognized, namely the cytoplasmically and the mitochondrially localized elements.
